Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation.
A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.
Vaandrager
A B
AB
Departments of Biochemistry, Cardiovascular Research Institute COEUR, 3000 DR Rotterdam, The Netherlands. Vaandrager@bc1.fgg.eur.nl
Smolenski
A
A
Tilly
B C
BC
Houtsmuller
A B
AB
Ehlert
E M
EM
Bot
A G
AG
Edixhoven
M
M
Boomaars
W E
WE
Lohmann
S M
SM
de Jonge
H R
HR
D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D018118:Chloride Channels / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D006801:Humans; D015640:Ion Channel Gating; D009226:Myristates; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins; D014162:Transfection
C505032:CFTR protein, human; D018118:Chloride Channels; D009226:Myristates; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017869:Cyclic GMP-Dependent Protein Kinases